Abstract

The molecular mechanisms determining the transmissibility and prevalence of drug-resistant tuberculosis in a population were investigated through whole-genome sequencing of 1,000 prospectively obtained patient isolates from Russia. Two-thirds belonged to the Beijing lineage, which was dominated by two homogeneous clades. Multidrug-resistant (MDR) genotypes were found in 48% of isolates overall and in 87% of the major clades. The most common rpoB mutation was associated with fitness-compensatory mutations in rpoA or rpoC, and a new intragenic compensatory substitution was identified. The proportion of MDR cases with extensively drug-resistant (XDR) tuberculosis was 16% overall, with 65% of MDR isolates harboring eis mutations, selected by kanamycin therapy, which may drive the expansion of strains with enhanced virulence. The combination of drug resistance and compensatory mutations displayed by the major clades confers clinical resistance without compromising fitness and transmissibility, showing that, in addition to weaknesses in the tuberculosis control program, biological factors drive the persistence and spread of MDR and XDR tuberculosis in Russia and beyond.

The location of Samara Oblast in Russia (red) and the Baltic States (Lithuania (Li), Latvia (Lv) and Estonia (E)) are shown in (a). The number of sequenced patient-isolates from each territory (green) and city (blue; Samara City (Sm), Togliatti (T) and Syzran (Sz)) of Samara Oblast (b) or district of Samara City (c) are shown inside circles. The area of each circle reflects coverage of the region (the number of isolates sequenced relative to the number of tuberculosis cases notified).

The four M. tuberculosis lineages: Beijing, CAS, EuroAmerican and EAI, are indicated. The EuroAmerican SNP-defined sublineages and the major Beijing clades are shaded. The ancestral node of the Beijing East European sublineage is indicated with a star. Radial dotted lines show the positions of isolates from the UK; those with an XDR phenotype are marked with white circles. The Estonian strain is indicated by a filled blue circle. The position of the reference sequence, H37Rv, is marked ‘R’. The East European sublineage, Clade A and Clade B had 100% bootstrap support ().

The phylogeny of 1,000 Russian isolates is depicted on the left; lineages are colored as . The first 16 columns depict drug resistance loci. ‘P’ denotes a promoter region. Within the 16S rRNA gene, rrsstr refers to the 530 stem-loop and 915 regions involved in streptomycin resistance and rrsinj to downstream regions associated with resistance to the second-line injectables. Colored bands represent different polymorphisms and include previously identified and novel mutations described in the text. The last three columns show nsSNPs in the RNA polymerase genes, rpoABC, excluding those shown in the RRDR. The genotypes illustrated are provided in full in .

The proportion of isolates harboring polymorphisms at each drug resistance locus was categorized by lineage. Asterisks indicate significant differences between lineages (). Data is based on the polymorphisms detailed in .

The number of isolates within clusters sharing a genotypic marker was estimated by maximum likelihood reconstruction of the polymorphisms onto the phylogeny. A cluster size of one suggests acquired resistance while larger clusters are indicative of primary transmitted resistance.